Download Wheat, Fusarium toxins and disease: the good, the bad and the ugly

Wheat, Fusarium toxins and disease: the good, the bad and the ugly
H. Y. (Michelle) Seow1, X. W. Guo2, W. G. Dilantha Fernando2
1
Department of Microbiology; 2 Department of Plant Science, University of Manitoba, Winnipeg, Manitoba
Impact Statement:
Statement: The more potent 3ADON toxintoxin-producing fusarium isolates are on the increase in Manitoba
INTRODUCTION
¾THE GOOD: Wheat is the most economically important
crop in Canada, with Manitoba exporting wheat to
approximately 66 different countries. It is processed into
flour, cereal food, animal feed and industrial products such
as ethanol.
¾THE BAD: Fusarium head blight (FHB) is the most
serious disease in wheat around the world. Fusarium
graminearum is the causal agent of FHB which lowers
wheat yield, damages grain quality, and causes animal feed
refusal and illness in humans by the mycotoxins it
produces- deoxynivalenol (DON), nivalenol (NIV), 3-acetyl
deoxynivalenol (3 ADON) and 15-actyl deoxynivalenol (15
ADON).
¾THE UGLY: Detection of less potent toxin 15 ADON has
been higher than the other toxins in Manitoba, but this
might be changing with the more potent 3 ADON
increasing and posing a danger to the industry.
OBJECTIVES
¾Obtain evidence that pathogen toxin profiles have changed
from less potent 15 ADON to more potent 3 ADON.
¾ Determine toxin profile of Fusarium graminearum strains
in Manitoba wheat fields.
RESULTS
MATERIALS AND METHODS
¾Wheat spikes of Superb and AC Barrie varieties were
collected from 31 fields in 2004 and 2005. These were
plated onto potato-dextrose agar (PDA) for 5 days.
¾Mycelia which had Fusarium graminearum
characteristics (red, pinkish, or orange) were transferred
onto synthetic nutrient agar for spore production and
single spored using water agar medium.
¾DNA extracted from mycelium of single spore culture.
Primer name
3CON
3NA
3D15A
3D3A
Sequence (5'-3')
TGGCAAAGACTGGTTCAC
GTGCACAGAATATACGAGC
ACTGACCCAAGCTGCCATC
CGCATTGGCTAACACATG
Fg16F
Fg16R
Sequence (5’-3’)
C D E
F G H I
← ~610 bp
M 1
2
3
← ~243 bp
Figure 2.Multiplex PCR products with TRI3 primers on 2% agarose gel.
Lanes. M- 1kb ladder. A, H, I- Fusarium graminearum isolates that produce
3 ADON. B,C, D, E, F, G- Fusarium graminearum isolates that produce 15
ADON.
CONCLUSIONS
RESULTS
4
Figure 1. Amplified Fusarium
graminearum specific PCR products on
2% agarose gel. Lanes. M- 1kb ladder.
1,3- Isolates that were not Fusarium
graminearum. 2,4- Isolates that were
Fusarium graminearum.
¾Results of multiplex PCR show that for all farms except
Hamiota, the percentage of Fusarium graminearum strains
that contain the gene which produces 15 ADON decreases
from 2004. Inversely, isolates which contain the gene that
produces 3 ADON increases from 2004 to 2005.
Results of multiplex PCR for 2004 and 2005 Fusarium graminearum
isolates from same location.
2005 Total
15 ADON
(%)
Portage la
Prairie
55.88
44.44
44.12
55.56
Size (bp)
Plumas
Hamiota
85.71
75
60
100
14.29
25
40
0
450
Rapid City
Virden
80
100
71.43
75
20
0
28.57
25
CTCCGGATATGTTGCGTCAA
GGTAGGTATCCGACATGGCAA
Size (bp)
840
610
243
2004 Total
15 ADON
(%)
¾Fusarium graminearum were detected using primer pairs
Fg16F and Fg16R specific to the species.
Primer name
Specificity
common to all
NIV specific
15ADON specific
3ADON specific
¾ For the 2004 samples, 131 isolates were confirmed to be
Fusarium graminearum through PCR where they had bands
around 450bp (Figure 1). Similarly, 121 isolates were
confirmed to be F. graminearum from the 2005 isolates.
Location
PCR Analysis- Species Detection
A B
TRI3 specific primers
←~450 bp
MATERIALS AND METHODS
Grain Samples, Isolation of Single Spores and
DNA Extraction
M
PCR Analysis- Mycotoxins
¾TRI3 specific primers were used in multiplex PCRs to detect
Nivalenol, 3 ADON and 15 ADON genes.
2004 Total 3 2005 Total 3
ADON (%) ADON (%)
¾Fusarium graminearum strains in Manitoba which
are able to produce 3 ADON toxins are increasing,
while those which are able to produce 15 ADON
strains are decreasing.
¾More studies should be done on this shift of 15
ADON to 3 ADON production, as well as on the
significance and impact of this shift on the wheat
industry to protect the future of the food and feed
industry.
ACKNOWLEDGMENTS
¾ The Natural Sciences and Engineering Research Council of Canada and
Canadian Wheat Board for the funding of this project.
¾ Daniel Brick for assistance in fieldwork.
¾ Todd Ward (Microbial Genomics and Bioprocessing Research Unit,
National Center for Agricultural Utilization Research, U.S. Department of
Agriculture, Preoria, IL) for TRI3 primer sequences.